Fig 2: RTN3 absence impairs ER tubules turnover but not macro-autophagy flux.(A) Western blot analysis of ER protein turnover in wild-type, Atg5–/–, Fip200–/–, Fam134b–/– and Rtn3–/– MEFs. Cells were starved with EBSS for the indicate time in the presence of 100 µM cicloheximide. SQSTM1 (p62) has been used as positive control for autophagy induction. (B) Western blot analysis of ER tubules markers in wild-type, Rtn3–/– MEFs and Rtn3–/– MEFs reconstituted with the human EGFP-RTN3L. (C) Western blot for GFP in Rtn3–/– MEFs transfected with human EGFP-RTN3L. (D,E) Western blot analysis of Lc3b and p62 in wild-type and Rtn3–/– MEFs. Cells were treated with 250 nM Torin1 (D) or EBSS (E) in the presence or absence of Bafilomycin A1, 200 ng/ml, for the indicated time. (F) Representative confocal imagines of wild-type and Rtn3 knockout MEFs, transfected with mCherry-EGFP-LC3B, in standard conditions (DMEM with 10% FBS) and after 6 hr EBSS treatment. Scale bars: 10 µm. (G) Quantification of LC3B positive autophagy puncta in wild-type and Rtn3–/– MEFs transfected with mCherry-EGFP-LC3B. Cells were grown in standard conditions or treated with EBSS for 2 hr. Number of cells >50 for each condition. Data are representative of three independent biological replicates. Error bars indicate s.d. No significant differences were detected between wild-type and Rtn3–/– MEFs.DOI: http://dx.doi.org/10.7554/eLife.25555.017

Fig 3: RTN3 interacts with the autophagy modifiers.(A) Venn diagrams of the interactors of the four RTNs. Numbers represent the identified peptides significantly enriched in three IP and mass spectrometry replicates for each RTN. (B) Annotation enrichment analysis of the interactors of long RTN1-4 isoforms. Bars represent the significantly enriched gene ontology biological process (GOBP), the gene ontology cellular components (GOCC), the gene ontology molecular function (GOMF), the over-expressed pathways (KEGG) and the domain enrichment (Pfam). The numeric value on the right side of the bar shows the Benjamini-Hochberg FDR value. (C) Scatter-plot for 1D annotation enrichment analysis of RTN3L interactor partners significantly enriched in three different IPs. (D) Volcano-plot for RTN3L SILAC-based interactome. Peptides with and Log2 Ratio H/L ≥1 and –Log10 p value > 1.3 are labeled in red. Three biological replicates were analyzed. (E) Co-IP of endogenous GABARAP and GABARAP-L1 with over-expressed long and short isoforms of RTN1-4. Over-expression was induced for 24 hr in U2OS TRex stable cell lines using 1 µg/ml of doxycycline. Bafilomycin A1 was added at the final concentration of 200 ng/ml for 2 hr. (F) Endogenous Co-IP of RTN3 with GABARAP in A549 cells. The ‘empty’ lane represents unconjugated beads. Bafilomycin A1, 200 ng/ml, was added for 2 hr.DOI: http://dx.doi.org/10.7554/eLife.25555.01910.7554/eLife.25555.020Figure 5—source data 1.IP-interactome of RTN1, RTN2, RTN3 and RTN4 long isoforms.IP-interactome analyses were performed using the SILAC-labeling strategy in U2OS after 24-hr treatment with 1 µg/ml of doxycycline. Bafilomycin A1, 200 ng/ml, was added for 2 hr. Peptides with Log2 (Heavy/Light [H/L]) ratios ≥1 and a p value ≤ 0.05 were considered significantly enriched.DOI: http://dx.doi.org/10.7554/eLife.25555.02010.7554/eLife.25555.021Figure 5—source data 2.IP-interactome of RTN1, RTN2, RTN3 and RTN4 short isoforms.IP-interactome analyses were performed using the SILAC labeling strategy in U2OS after 24-hr treatment with 1 µg/ml of doxycycline. Bafilomycin A1, 200 ng/ml, was added for 2 hr. Peptides with Log2 (Heavy/Light [H/L]) ratios ≥ 1 and a p value ≤ 0.05 were considered significantly enriched.DOI: http://dx.doi.org/10.7554/eLife.25555.021

Fig 4: RTN3 over-expression induces ER tubules fragmentation during starvation.(A, B) Schematic representation of the protein structure and topology of FAM134B (A) and RTN3 long and short isoforms (B). (C) Immunofluorescence of HA and LC3B in U2OS TRex stable cell lines expressing FLAG-HA-RTN3L after 24 hr treatment with 1 µg/ml of doxycycline. Cells were kept in standard growing condition (DMEM with 10% FBS) or starved with EBSS for 6 hr. RTN3L was monitored using anti HA antibody, while autophagy induction was visualized using anti-LC3B antibody. Bafilomycin A1 was added at a final concentration of 200 ng/ml. Scale bars: 10 µm. (D) Quantification of U2OS TRex FLAG-HA-RTN1-4L cells with ER tubule fragment after 6 hr starvation with EBSS plus Bafilomycin A1, 200 ng/ml. Number of cells >100 for each condition. Data are representative of three independent biological replicates. Error bars indicate s.d. (E) Super-resolution fluorescence microscopy (dSTORM) of ER fragments in U2OS TRex FLAG-HA-RTN3L cells stained with anti HA and anti LC3B antibodies after 6 hr starvation with EBSS plus Bafilomycin A1, 200 ng/ml. Scale bar: 0.5 µm. (F) Schematic representation of ER tubules fragmentation and LC3 labeling in U2OS TRex FLAG-HA-RTN3L cells. The red square indicates the level of high resolution represented in panel E.DOI: http://dx.doi.org/10.7554/eLife.25555.002

Fig 5: Autophagy machinery influences RTN3 ability to fragment ER tubules.(A) Venn diagrams of the interacting partners for RTN3L and RTNS. Numbers represent the identified proteins significantly enriched in three IP and mass spectrometry replicates for each isoform. (B) Schematic representation of the common and unique significantly enriched peptides for RTN3L and RTN3S. (C) Volcano-plot for RTN3L SILAC-based interactome. The interactors partners of RTN3L with and Log2 Ratio H/L >1 and –Log10 p value > 1.3 are labeled in dark blue. The common peptides between RTN3L and RTN3S, with and Log2 Ratio H/L >1 and –Log10 p value > 1.3, are labeled in red. Data represent three independent biological replicates. (D) Western blot analysis of ER tubules markers in wild-type, Rtn3–/– MEFs and Rtn3–/– MEFs reconstituted with the human EGFP-RTN3S or EGFP-RTN3L∆6LIRs. (E) Western blot for GFP in Rtn3–/– MEFs transfected with human EGFP-RTN3S or EGFP-RTN3L∆6LIRs. (F) Immunofluorescence of HA and LC3B in U2OS TRex FLAG-HA-RTN3L; FAM134B or ATG7 knockout cells induced for 24 hr with 1 µg/ml of doxycycline and starved for 6 hr with EBSS plus Bafilomycin A1, 200 ng/ml. RTN3L was monitored using anti HA antibody, while autophagy induction was visualized using anti-LC3B antibody. Scale bars: 10 µm. (G) Quantification of cells presenting ER tubule fragments after 6 hr starvation with EBSS plus Bafilomycin A1, 200 ng/ml. Number of cells >500 for each condition. Data are representative of three independent biological replicates. *p<0.01. Error bars indicate s.d. (H) Western blot analysis for ATG7 and FAM134B protein level in U2OS TRex FLAG-HA-RTN3L cells after ATG7 or FAM134B gene knockout by CRISPR-CAS9 technology.DOI: http://dx.doi.org/10.7554/eLife.25555.02910.7554/eLife.25555.030Figure 7—source data 1.Comparison of the IP-interactome of RTN3L and RTN3S.Comparison of the IP-interactomes analyses were performed using the SILAC-labeling strategy in U2OS after 24 hr treatment with 1 µg/ml of doxycycline. Bafilomycin A1, 200 ng/ml, was added for 2 hr. Peptides with Log2 (Heavy/Light [H/L]) ratios ≥1 and a p value ≤ 0.05 were considered significantly enriched.DOI: http://dx.doi.org/10.7554/eLife.25555.030